EP2259049A1 - Method and measuring device for qualitative and quantitative analysis of bodily fluids - Google Patents

Abstract

The method involves tempering a body fluid sample (5) in a cryostat (3) at certain temperature range. The body fluid sample is irradiated and simulated by light in the ultraviolet, visible or infrared region. The cryostat satisfies both the function of the temperature control of the body fluid sample and the function of a sample holder and a sample cell. An independent claim is also included for a measuring device for qualitative and quantitative analysis of body fluids, particularly blood, urine or sweat.

Description

The present invention relates to a method and a measuring device for the qualitative and quantitative analysis of body fluids, in particular blood, sweat and urine.

• der gemessene Blutzuckerwert ein erhöhter Glukosespiegel ist,
• gleichzeitig weitere Indikatoren, wie zum Beispiel das Vorliegen von diabetischer Dyslipidämie, das heißt erhöhten Blutfettwerten, bestimmt werden.

Stark erhöhte Blutfettwerte bei Vorliegen einer Hyperlipidämie tragen zu Bluthochdruck, Herzerkrankung und Schlaganfall bei, wie in der US 6,127,138 A beschrieben. Deshalb wird zur Diagnose von kardiovaskulären Krankheiten, wie beispielsweise konorarer Herzerkrankung, routinemäßig der Blutcholesterinspiegel analysiert. Dabei werden der Gesamtcholesteringehalt sowie die Plasmatriglyceridspiegel gemessen. Zudem werden oftmals die Spiegel der Haupt-Lipoprotein-Bestandteile von Cholesterin untersucht. Eine Methode, welche es erlaubt, mehrere verschiedene Blutbestandteile parallel nebeneinander zu untersuchen, wird in den US-Patentschriften US 4,933,844 A und US 5,343,389 A dargestellt. Dabei sollen die verschiedenen Bestandteile von Blutplasma und Blutserum mit Hilfe der ¹H-NMR-Spektroskopie auf Konzentration bestimmt werden. Vorteil dieses Verfahrens ist neben der parallelen Analyse mehrerer Bestandteile die hohe Empfindlichkeit der NMR-Analyse bezogen auf die Blutbestandteile, wie aus der DE 600 26 287 T2 hervorgeht. Als nachteilig sind hier die Komplexität und Kostenintensität des NMR-Gerätes zu nennen. Diese Kriterien bewirken, dass es für Allgemeinmediziner nicht oder kaum möglich ist, Blutuntersuchungen, die eine frühzeitige Erkennung von Diabetes und Hyperlipidämie zulassen, vor Ort, das heißt in der eigenen Praxis, durchzuführen. Die US 5,341,805 A beschreibt eine Methode zur Bestimmung der Glukosekonzentration in Blut mit Hilfe der Fluoreszenzspektroskopie. Bei dieser Methode wird das Blut mit Licht der Wellenlänge 308 nm angeregt und die resultierende Fluoreszenz wird spektral zerlegt aufgetragen. Aufgrund des Verhältnisses der charakteristischen Glukosebanden im Spektrum sollen hier Rückschlüsse auf die Konzentration der Glukose im Blut bestimmt werden. Die Sensitivität dieser Messung, bezogen auf die Glukosekonzentration, lässt jedoch sehr zu wünschen übrig. Der Nachweis von weiteren wichtigen Indikatoren, wie beispielsweise den Fettanteilen, ist mit dieser Methode jedoch nicht bekannt.The chemical composition of body fluids, especially of blood, sweat and urine and their change in response to various environmental influences are in today's medicine important characteristics for the diagnosis of various diseases. Among other things, in the context of the increasing frequency of obesity worldwide and the associated increase in the likelihood of developing serious illnesses such as diabetes, heart attack and other cardiovascular diseases, a comprehensive, highly sensitive and simple method of examining various constituents of body fluids is emerging , in particular of blood components, which indicate these diseases early, increasingly in the spotlight. Diabetes mellitus alone has become a worldwide widespread disease. The "International Diabetes Ferderation" speaks of the epidemic of the 21st century (Diabetes out of control, press release of 04 December 2006). According to the UN Resolution on Diabetes ( www.unitefordiabetes.org/campaign/resolution.html ), diabetes mellitus is the only non- communicable disease on a level with infectious diseases such as tuberculosis, malaria or HIV. It is currently estimated that about 246 million people worldwide have diabetes. According to initial forecast estimates, this number is expected to increase to 380 million by 2025. Given the dramatic increase in diabetes and the inability to cure diabetes, the only thing that remains is to step up efforts to detect early type 2 diabetes, and thus the disease to prevent its formation. However, in order to be able to prevent diabetes in good time, it is necessary to identify persons with an increased risk of disease at the diagnostic-free interval or beforehand. As a pragmatic approach, the FINDRISC (FINish Diabetes Rlsc SCore) Risk Questionnaire (according to Lindstrom J. and Tuomilehto J .: The Diabetes Risk Score. Diabetes Care, 2003; 26 (3): 725-731 ) . However, this approach only provides risk estimates for the disease and no medical details. Medically, type 2 diabetes is traditionally diagnosed by the detection of elevated blood glucose levels, hyperglycemia. Conventional tests for type 2 diabetes, however, identify a change in blood sugar levels only in a very advanced state of the disease. The detection of subhyperglycemic glucose levels is not sufficient here, without the aid of further diabetes type 2 indicators, in order to conclude that the patient has an increased risk of developing the disease. Early detection of the increased risk of developing insulin resistance or type 2 diabetes is thus possible when

• the measured blood glucose value is an elevated glucose level,
• at the same time other indicators, such as the presence of diabetic dyslipidemia, ie elevated blood lipid levels, are determined.

Highly elevated blood lipid levels in the presence of hyperlipidemia contribute to high blood pressure, heart disease and stroke, as in the US 6,127,138 A described. Therefore, blood cholesterol levels are routinely analyzed for the diagnosis of cardiovascular diseases, such as coronary heart disease. The total cholesterol content and the plasma triglyceride levels are measured. In addition, the levels of the major lipoprotein components of cholesterol are often examined. A method that allows several different blood components in parallel Side by side, US Pat US 4,933,844 A and US 5,343,389 A shown. The different constituents of blood plasma and blood serum are to be determined for concentration by means of ¹ H-NMR spectroscopy. Advantage of this method, in addition to the parallel analysis of several components, the high sensitivity of the NMR analysis based on the blood components, as from DE 600 26 287 T2 evident. The disadvantage here is the complexity and cost intensity of the NMR device. These criteria mean that it is not or hardly possible for general practitioners to carry out blood tests, which allow early detection of diabetes and hyperlipidemia, locally, ie in their own practice. The US 5,341,805 A describes a method for determining the glucose concentration in blood by means of fluorescence spectroscopy. In this method, the blood is excited with light of wavelength 308 nm and the resulting fluorescence is spectrally decomposed applied. Based on the ratio of the characteristic glucose bands in the spectrum conclusions about the concentration of glucose in the blood should be determined. However, the sensitivity of this measurement, based on the glucose concentration, leaves much to be desired. However, the detection of other important indicators, such as fat levels, is not known using this method.

Extensive blood analyzes, which give a precise overview of the chemical and quantitative composition of the blood, are time-consuming and usually very expensive. Quick blood tests that are easier to handle and also significantly cheaper, usually only provide analysis data on a blood component, such as the lactate test or the glucose test. For example, the patient can only be tested for diabetes in diagnostic diagnostics with the aid of the rapid medical test. Scientifically, a diagnosis of diabetes is made only because of increased blood sugar levels was, but extremely doubtful. A reliable diagnosis requires proof of at least one other disease indicator. In addition, most diabetes diagnostic methods determine a change in blood sugar only in a strong increase in blood sugar, the so-called hyperglycemia. However, hyperglycemia is a very late stage in the chain of events leading to a full-blown diabetes. Thus, it is hardly possible to prevent the onset of diabetes type 2 for patients with such hyperglycaemia. On the other hand, methods that detect an increase in blood sugar levels at an earlier stage often do not allow a clear assessment of the risk of disease. These methods require proof of another disease indicator. However, such techniques, which allow the detection of multiple disease indicators simultaneously, are either extremely time consuming or expensive.

The object underlying the invention is to provide a method which makes it possible to analyze several components of a body fluid, in particular the blood, sweat and urine in parallel. The aim is to measure both the concentration of the respective substance in the body fluid and its concentration change as a function of various environmental factors acting on the test person. In addition to this quantity-based analysis, the method developed should provide the opportunity to visualize and analyze the chemistry and chemical changes associated with exposure to the constituents of each bodily fluid. The analytical data obtained for the individual components can be used to make statements about various diseases, such as diabetes or mitochondrial dysfunctions.

The object of the invention is achieved by a method for the qualitative and quantitative analysis of body fluids, in particular of blood, urine or sweat. In this case, fluorescence spectroscopic methods are used to study the body fluids. In the first process step a), a temperature control of the body fluid sample takes place at temperatures between 200 K and 4 K in a cryostat. In the second step b) irradiation and excitation of the body fluid sample with light in the UV, visible or infrared range at temperatures between 200 K and 4 K is performed. The cryostat fulfills both the function of temperature control of the body fluid sample and the function of a sample holder and a sample cell by the sample cell is arranged in the cryostat and completed by a Kryostatenwandung outwards, the sample cell contacted directly with a heat exchanger of the cryostat thermally. The fluorescence radiation produced during excitation of the body fluid sample is spectrally decomposed into individual fluorescence signals in method step c), which are subsequently detected in method step d). Thereafter, in process step e), a comparison of the fluorescence signals obtained and of corresponding comparison data takes place. This comparison then allows an evaluation of the fluorescence signals.

The method described here for the qualitative and quantitative analysis of body fluid constituents is suitable for both human and veterinary uses. The invention is based on the property of substances to absorb light of specific wavelengths and to re-emit them after internal conversion in the form of fluorescence. The resulting fluorescence spectra are characteristic and, taking into account band positions in the spectrum and the fluorescence lifetime, can be used for chemical sample analysis. The property of a substance to fluoresce is strong depending on how and with which environment molecules the fluorescent substance interacts. Such interactions suppress the intensity of fluorescence and can lead to complete quenching of fluorescence. The two most important types of quenching processes are dynamic quenching and static quenching. In the dynamic, ie burst-based fluorescence quenching the excited molecule is transferred by energy exchange with the quencher radiationless in the ground state, whereas in the statistical state quenching of the dye molecule and the quencher in the ground state, a complex is formed which does not fluoresce in the excited state, but also radiationless returns to the ground state. Thus, to increase the fluorescence intensity of the fluorophore, it is necessary to minimize the quenching effects prevailing under normal conditions. According to the invention, this is done by the significant reduction in the temperature in the body fluid sample to be examined. This reduction in temperature causes the particle mobility of the fluorophore and quencher within the body fluid sample to decrease, thus resulting in increased fluorescence intensity.

When examining blood samples, a well evaluable fluorescence signal could be measured for "Low Density Lipoprotein" (LDL) even from a temperature of 200 K, which did not change significantly even if the temperature were further reduced. Preferably, however, the temperature control of the body fluid sample in process step a) to temperatures between 150 K and 4 K, since there provide most components of body fluid samples well evaluable fluorescence signals. Temperatures between 100 K and 10 K proved to be particularly advantageous.

In one embodiment of the method according to the invention, the irradiation and excitation in step b) with light of wavelength 266 nm, preferably laser light, since most organic compounds have a strong absorption in the UV range. However, various organic compounds also have excitation maxima in the vis range. An example of this is the fluorescence of flavin adenine dinucleotide (FAD). This substance can be excited to fluoresce even at room temperature with an excitation wavelength of 450 nm and emitted in the range of 515 nm. Also with this substance, a significant increase in the intensity of the emission signal could be determined with a reduction in temperature. In addition, it was observed in low-temperature glucose studies that pulsed laser excitation with a femtosecond laser at 700 nm (IR range) also produced glucose fluorescence. However, this fluorescence is more likely to be associated with multiphoton excitation.

However, the irradiation of the body fluid sample in method step b) can also take place simultaneously with several different wavelengths. Alternatively, there is also the possibility of a successive irradiation with different wavelengths. This fluorescence of various components of the body fluid can be stimulated both simultaneously and sequentially.

A particular advantage of the invention is to be considered that under given circumstances, the detection according to process step d) and thus the measurement of the fluorescence signals of the various ingredients of the respective body fluid can be performed simultaneously. Thus, in the analysis of blood constituents, at least two of the indicators may include blood sugar levels, insulin levels, levels of lipoproteins, levels of homocysteine, long-term blood glucose (HbA1c), and fibrinogen levels be examined simultaneously. In particular, in the study of diabetes type 2, the measurement of the blood glucose level can be carried out simultaneously with the measurement of blood protein values. In another application in connection with the analysis of blood components, the use of the method in the detection of mitochondrial dysfunction, for the determination of the lactate / pyruvate ratio, the fluorescence signals of both components, lactate and pyruvate, can be measured simultaneously. In the analysis of urinary components in conjunction with the study on type 2 diabetes, preferably the measurements of the sugar content, the content of acetone and / or albumin are carried out simultaneously.

The excitation of the frozen sample is carried out according to an embodiment of the method according to the invention with light of wavelength 266 nm. Occasionally be evaluated at a measurement temperature of 200 K evaluable fluorescences, the optimum cooling temperature between 150 K and 4 K. In one embodiment of the invention, when examining the lactate value in the blood, a clearly evaluable spectrum for lactate can already be recognized by reducing the temperature to 200 K, which increases again as the temperature is further reduced to 100 K and 10 K.

For the comparison of the fluorescence data obtained and the comparison data in step e) as comparison characteristics are preferably the intensity of the fluorescence, the area below a fluorescence band, the position of the individual fluorescence band maxima in the total spectrum, the ratio of different band maxima within a spectrum to each other, the quantum yield of the sample and the lifetime of the fluorescence of each fluorophore provided. In this case, the measured value of the desired body fluid constituent determined by low temperature is preferably compared with a predetermined test criterion. For example, a patient to be examined Accordingly, as ill or identified as a risk patient, when several of the measured body fluid constituents have changes from the predetermined test criterion. In general, it is advantageous if the test and reference criterion has previously been verified by measurements independent of this method.

• eine Lichtquelle für die Anregung der Körperflüssigkeitsprobe,
• einen Kryostaten für das Temperieren der Körperflüssigkeitsprobe,
• einen Fluoreszenzspektrographen für das Zerlegen der gewonnenen Fluoreszenzstrahlung der Körperflüssigkeitsprobe,
• einen Bildgebungsdetektor,
• eine Probenmesszelle, die gleichzeitig als Probenhalterung dient und im Kryostat angeordnet ist, wobei sie durch eine Kryostatenwandung nach außen hin abgeschlossen ist und direkt mit einem Wärmeübertrager des Kryostaten thermisch kontaktiert, und
• eine UV-gängige, temperaturstabile Küvette, mit der die Körperflüssigkeitsprobe in die Probenmesszelle eingeführt wird.

For carrying out the method described above, a measuring device is used which comprises the following components:

• a light source for stimulating the body fluid sample,
• a cryostat for tempering the body fluid sample,
• a fluorescence spectrograph for decomposing the obtained fluorescence radiation of the body fluid sample,
• an imaging detector,
• a sample measuring cell, which simultaneously serves as a sample holder and is arranged in the cryostat, wherein it is closed by a Kryostatenwandung outwardly and thermally contacted directly with a heat exchanger of the cryostat, and
• a UV-compatible, temperature-stable cuvette, with which the body fluid sample is introduced into the sample cell.

In a preferred embodiment of the invention, the light source, the body fluid sample thermostating cryostat, the fluorescence spectrograph, and the imaging detector are within one arranged compact measuring device. According to the invention, the sample measuring cell, which also serves as a sample holder, is arranged in the cryostat. The cryostat thus fulfills both the task of temperature control of the body fluid sample and the task of the sample measuring cell. The cryostat wall, which closes off the sample measuring cell to the outside and thus prevents heat exchange with the light source and the fluorescence spectrograph, is preferably formed by at least two UV-common windows offset by 90 ° from each other. In an alternative embodiment of the invention, the light source, the cryostat for deep-freezing the body fluid sample, the fluorescence spectrograph and the imaging detector act as a sum of individual devices with each other.

The light source used to excite the frozen body fluid sample is, in an advantageous embodiment, a broadband light source. As broadband light sources in particular mercury vapor lamps, xenon high-pressure lamps or deuterium lamps are provided. In an alternative embodiment of the measuring device according to the invention, a narrow-band light source can be used for the excitation of the frozen body fluid sample. In this case, in particular light-emitting diode (LED) light sources or laser light sources come into question, wherein in the measuring device a plurality of light sources of different wavelengths can be provided.

For sample cooling or for tempering basically any type of cryostat can be used, with the handling of the overall system and the economy of cryostat with closed refrigerant circuit is better compared to those with open refrigerant circuit. Preferably, the cryostats used are refrigerator cryostats, evaporator cryostats, pulse tube cryostats or sterling coolers used. As a refrigerant, for example, helium or nitrogen can act, and due to the required very low temperatures, the use of helium is considered preferable. At the same time, however, the use of refrigerant mixtures is conceivable. Preferably, the sample cell is directly connected to a heat exchanger of the cryostat and a heater for regulating the temperature of the body fluid sample.

The obtained data of the Kryofluoreszenzmessung be spectrally dissected according to a preferred embodiment of the method according to the invention and then detected by means of an imaging detector. In this case, various photomultipliers (PMT), pin photodiodes, avalanche photodiodes or charge coupled device (CCD) cameras can be used as the imaging detector.

The advantages of the measuring device according to the invention are, above all, that it is suitable both for human and for veterinary uses. Furthermore, with this measuring device and the measuring method, the analysis of several components in parallel in only one device is possible. Thus, there is also the possibility of analyzing different clinical pictures by means of only one method and one device.

The high sensitivity of the method allows for early detection of changes. Last but not least, the method is also cost-effective in comparison with previously developed methods of similar efficiency. Another advantage is the ease of use of the method with closed refrigerant circuit. The method allows both the quantitative and the chemical analysis of the individual blood components. Prior treatment of the blood sample (sample preparation) is only conditionally necessary. This contributes as much to the time savings as the fact that the Assay results are available relatively quickly. In addition, no chemical change of the blood is necessary. Overall, all the above-mentioned advantages contribute to the fact that the method is also considered to be very economical over a longer period of time and at high frequency with the refrigerant circuit closed.

Fig. 1a:

eine vereinfachte Darstellung der Messvorrichtung,

Fig. 1b:

ein vereinfachtes Prinzip der Messvorrichtung für Messungen bei Raumtemperatur und bei tiefen Temperaturen,

Fig. 1c:

die Messvorrichtung mit Kompressor und Auswertungseinheit,

Fig.2:

die Fluoreszenzantwort von Glukose bei Bestrahlung mit Laserlicht der Wellenlänge 266 nm unter variierenden Temperaturbedingungen,

Fig. 3:

die Fluoreszenzantwort von "Low Density Lipoprotein" (LDL) bei Bestrahlung mit Laserlicht der Wellenlänge 266 nm unter variierenden Temperaturbedingungen,

Fig. 4:

die Fluoreszenzantwort von Laktat bei Bestrahlung mit Laserlicht der Wellenlänge 266 nm unter variierenden Temperaturbedingungen,

Fig. 5:

die Fluoreszenzantwort von Pyruvat bei Bestrahlung mit Laserlicht der Wellenlänge 266 nm unter variierenden Temperaturbedingungen.

Further details, features and advantages of the invention will become apparent from the following description of the embodiments I to III with reference to the accompanying drawings. Show it:

Fig. 1a:

a simplified representation of the measuring device,

Fig. 1b:

a simplified principle of the measuring device for measurements at room temperature and at low temperatures,

Fig. 1c:

the measuring device with compressor and evaluation unit,

Figure 2:

the fluorescence response of glucose when irradiated with laser light of wavelength 266 nm under varying temperature conditions,

3:

the fluorescence response of "low density lipoprotein" (LDL) when irradiated with laser light of wavelength 266 nm under varying temperature conditions,

4:

the fluorescence response of lactate when irradiated with laser light of wavelength 266 nm under varying temperature conditions,

Fig. 5:

the fluorescence response of pyruvate upon irradiation with laser light of wavelength 266 nm under varying temperature conditions.

The Fig. 1a shows a simplified representation of the measuring device 1. The Fig. 1b shows a simplified principle of the measuring device 1 for measurements at room temperature and at low temperatures. In order to examine a body fluid sample with the method described here, in a first step, approximately two milliliters of the body fluid to be examined must be taken from the patient. In the following, this body fluid sample 5 is placed in a UV-compatible temperature-stable cuvette 6. The cuvette 6 and its contents are brought into a combined apparatus, essentially consisting of a light source 2, a cryostat 3 and a fluorescence spectrograph with an imaging detector 4. It is in the FIGS. 1a . 1b and 1c only one possible arrangement shown.

As the light source 2 for the respective investigations depending on the desired objective broadband light sources 2, such as mercury vapor lamps, xenon high pressure lamps, deuterium lamps or narrow-band light sources 2, such as LEDs and lasers (Titanium: Sapphire, diode laser, dye laser others). Both types of light sources 2 offer their advantages for different applications. Broadband light sources 2, because of their multiplicity of wavelengths, provide a better opportunity to simultaneously detect many different constituents of the body fluid sample 5. Narrow-band light sources 2 can be used specifically for excitation of specific sample components 5 due to the light emission over a small wavelength range. The disadvantage of using this narrowband However, light sources 2 is that due to various absorption maxima of the contents of the body fluid sample 5 with such a light source 2, not all the ingredients are excited to fluoresce. Increasing the diversity of the substances excited with a narrow-band light source 2 within the body fluid sample 5 can be due to the increase in the incident light energy, as it is for example provided by the use of lasers. However, in order to be able to cover the entire spectrum of possible fluorescences within the body fluid sample 5, the measuring device 1 must be equipped with multiple light sources of different wavelengths even when using lasers.

In the present arrangement, the cryostat 3 fulfills both the function of temperature control of the body fluid sample 5 and the function of the sample holder 7 and the sample measuring cell 7. After introduction of the UV-standard temperature-stable cuvette 6 in the sample holder 7, which directly with the heat exchanger 8 and a Heating 9 is connected to regulate the temperature of the body fluid sample 5, the body fluid sample 5 is cooled to a desired examination temperature and tempered at this temperature for some time, depending on the nature of the sample. Various measurements show that the optimal cooling temperature is between 150 K and 4 K, and occasionally, as in the case of "low density lipoprotein" (LDL), detectable fluorescences can already be detected in a blood sample at 200 K. The cryostat wall 10, which closes off the sample measuring cell 7 towards the outside and thus prevents heat exchange with the light source 2 and the fluorescence spectrograph 4, is characterized by at least two UV-common windows 11, for example Suprasil® quartz glass, offset by 90 °.
For sample cooling, for the method described any kind of known refrigerators, for example, based on the

Evaporator technology, refrigerator technology or pulse tube technology work, are used, with the handling of the overall system and the economy of cryostat 3 with closed refrigerant circuit is better compared to those with open refrigerant circuit. Helium, nitrogen or mixtures of refrigerants can act as refrigerants, whereby the use of helium is considered to be preferred due to the very low temperatures required. The refrigerant can, as in Fig. 1c represented, with the help of various pumps and compressors 12 are transported via a flexible hose system 13 to the heat exchanger 8. The body fluid sample 5, which is temperature-controlled to the examination temperature in the sample chamber, is then illuminated within the cryostat 3 with the aid of the light source 2 via one of the UV-standard windows 11. The fluorescence emissions of the sample constituents 5 generated by the irradiation are led out of the sample chamber via one of the further windows, spectrally decomposed in the fluorescence spectrograph 4 and subsequently detected. In this case, various photomultipliers (PMT), pin photodiodes, avalanche photodiodes or charge coupled device (CCD) cameras can be used as the imaging detector 4.
The evaluation of the fluorescence data thus obtained is carried out with the aid of special software via a computer 14 as an evaluation unit 14. With the help of this software, it is possible to normalize the data obtained, numerically and graphically represent their most important characteristics and then evaluate with a database of values for healthy subjects in the normal state (that is: no form of stress) to compare. Both static fluorescence data and dynamic fluorescence data are compared. As important comparison characteristics are the intensity of the fluorescence, the area below a fluorescence band, the position of the individual fluorescence band maxima in the total spectrum, the ratio of different band maxima within a spectrum to each other, the quantum yield the body fluid sample 5 and the lifetime of the fluorescence (time domain, frequency domain). Changes in intensity, for example, indicate disturbances in the metabolism. Shifts in emission maxima and changes in fluorescence lifetimes indicate different chemical environments of the fluorophore within the body fluid sample 5. Based on the different fluorescence lifetimes, it is also possible with this evaluation software to differentiate fluorescences, which have changed due to chemical bonding, according to the influence of the respective binding partner. This can play an important role, especially in such studies, where it is crucial how much foreign substance is associated with the substance to be analyzed (see example diabetes).

I. Use in the early detection of diabetes and its sequelae

As already described in the statements on the state of the art, diabetes type 2 and its accompanying and sequelae represent an important and increasingly occurring health risk in everyday life of humans. A method for simply determining whether a subject is a person Diabetes type 2 risk patients is not yet given in the current state of the art. With the present invention, a way is to be given to recognize the risk of the expression of a patient for diabetes type 2 in good time and to initiate preventive measures to prevent the disease and its accompanying and sequelae.

To determine the type 2 diabetes risk with the present method, the subject may be a person suspected of having an increased type 2 diabetes risk, a person who already has diabetes type 2 symptoms to be an asymptomatic person. The examiner has to remove about two milliliters of body fluid from the patient become. This may be in the form of, for example, blood, urine, and / or sweat, and blood should preferably be used for the present method. Subsequently, the taken body fluid sample of the body fluid is examined for various signs of insulin resistance. Insulin resistance is the failure of the body to respond normally to insulin. Insulin resistance syndrome is often a precursor to type 2 diabetes and refers to an amount of insulin resistance-related medical conditions in which high blood sugar levels stimulate the production of insulin. If the patient is no longer able to process excess insulin normally, insulin levels increase. As a result, the patient eventually has high blood sugar levels (hypergainemia) and high levels of insulin (hyperinsulemia). Such conditions cause insulin to lose its ability to regulate fat metabolism. Resulting excess fats enter the bloodstream (hyperlipidemia) and contribute to hypertension, heart disease and stroke. The body fluid sample can thus be assayed for various substances which are suitable as typical indicators of increased risk of diabetes type 2 disease. In the blood, for example, studies of blood sugar level, insulin level, lipoproteins (LDL = high density lipoproteins, lipoprotein a), the level of homocysteine, long-term blood glucose (HbA1c) and fibrinogen levels as risk studies feasible. In such an investigation, urine could also be tested for sugar content, but also for other factors such as acetone or albumin (microalbuminuria).

The sampled body fluid sample 5 is significantly cooled down in the following step (temperatures between 200 K and 4 K), irradiated with light of suitable wavelength and the resulting fluorescence spectrum is added. As an example, the fluorescence spectra of the relevant diabetes type 2 indicators glucose and low density lipoprotein [LDL] generated according to the above procedure may be mentioned in this document. Both indicators were investigated within the embodiment when irradiated with laser light of wavelength 266 nm under varying temperature conditions. In the Figures 2 and 3 Spectra of relevant diabetes type 2 indicators (glucose spectrum, Low Density Lipoprotein spectrum [LDL]) are shown at different temperatures and conditions alike. It can be seen that the intensity of the fluorescence signal for both substances in Fig. 2 and Fig. 3 with decreasing temperature rises. For both indicators, the framework conditions during the measurements were kept constant. Only the examination temperature was varied. Glucose shows according to Fig. 2 under selected conditions no fluorescence at room temperature. At a body fluid sample temperature of 100 K, a clear fluorescence signal of the glucose could be observed, which increased in intensity and structural properties as the temperature was further reduced to 10K. Thus, even at temperatures of about 100 K an evaluable glucose spectrum could be obtained, but due to the significantly better fine structuring of the spectrum, the measurement at 10 K is easier to interpret. Also, "Low Density Lipoprotein" [LDL] showed, as in Fig. 3 can be seen at room temperature under the chosen conditions no fluorescence response. With decreasing temperature, however, a clearly interpretable fluorescence signal was shown. In contrast to the studies on glucose, for low density lipoprotein [LDL] a well evaluable fluorescence signal could be measured already from a temperature of 200 K, which did not change significantly even with further reduction of the temperature. In addition to the in the Figures 2 and 3 shown intensity versus wavelength, the lifetime of fluorescence is an important criterion for the characterization of the individual investigated indicator substances. The fluorescence lifetime is also specific and strongly dependent on the ambient temperature during the measurement. For the evaluation, the obtained data of the respective indicators are normalized after the measurement and their characteristic values are compared with tabulated values of healthy, sub-hyperglycemic patients by means of an evaluation software. In addition to the general comparison criteria, the study of diabetes type 2 indicators, including the different fluorescence lifetimes, makes it possible to separate different chemical environments (such as different glycation levels of hemoglobin [HbA1 c value]) of the substance to be studied and to characterize.

The present invention thus provides the analytical basis for the early detection of the risk of developing type 2 diabetes, in that a fluorescence spectroscopic analysis test for glucose levels simultaneously with one or more fluorescence spectroscopic analysis based assays for assessing other risk factors, for example blood protein levels , can be carried out. Depending on the design of the measuring device 1, it is thus possible with this method to characterize and evaluate at least two but also significantly more relevant diabetes type 2 indicators within a sample and within one measurement cycle. Due to the fact that the detection of only one of the indicative parameters can usually also be influenced by other environmental processes, the possibility of detecting various parameters presented here represents a significant improvement in the risk assessment.

II. Lactate determination in performance diagnostics

Interesting results can also be obtained in the field of sports performance diagnostics by determining the lactate values with the aid of this method. Lactate is the salt of lactic acid and is the end product of anaerobic glucose metabolism. If the body does not have enough oxygen for aerobic metabolism due to different stress situations, energy is provided by anaerobic glycolysis, which produces lactic acid as a degradation product Enriching blood. This accumulation causes an increase in the blood lactate value. Increased lactate levels are therefore usually an indication that individual areas of the body are insufficiently supplied with oxygen. For this reason, lactate is the most important indicator for the assessment of physical performance. The determination of the blood lactate value is used in performance and mass sports for training control. During exercise, the lactate concentration in the blood increases. By creating a lactation curve (lactate concentration depending on the stress level), the training state of an athlete can be assessed. The later the lactate value increases due to the stress, the better the training state of the subject. To determine the lactate concentration in the blood of the subject using the present method, this must be taken about two milliliters of blood. The withdrawn blood sample 5 is then cooled down to temperatures between 200 K and 4 K and irradiated with light of suitable wavelength. Subsequently, the resulting fluorescence spectrum is recorded.

Fig. 4 shows, by way of example, the fluorescence of lactate when excited with 266 nm wavelength laser light under varying temperature conditions. It can be clearly seen from the maxima of the various spectra that the sensitivity of the detection to lactate is markedly increased by the use of the method of cooling down the sample 5 described here. at Measurements under room temperature conditions could not measure autofluorescence of the lactate for the settings chosen in this experiment. With a reduction of the temperature to 200 K, a clearly evaluable spectrum for lactate can already be seen, which increases again with further reduction of the temperature to 100 K and 10 K. Of the Fig. 4 It can be seen that by lowering the temperature from 200 K to 100 K a very large influence on the fluorescence intensity prevails, whereas the reduction from 100 K to 10 K has a comparatively less influence on the intensity. The obtained fluorescence data of the lactate are normalized and their characteristic data, such as the position of the band maxima, intensity of the fluorescence, ratio of different band maxima to each other and the lifetime of the fluorescence, compared with tabulated values before the load and evaluated.

III. Use in the detection of mitochondrial dysfunction

Mitochondrial dysfunctions are one of the main causes of human, neurodegenerative and neurometabolic diseases. Mitochondrial diseases (mitochondropathy) are, in the narrower sense, all disorders of enzymes that are involved in the energy production of cells summarized. Damaged mitochondria produce nitric oxide. This nitric oxide inhibits energy production in the mitochondria, across organs. The affected cells are thus in a chronic energy deficit, are thus more easily exhaustible and require longer rest periods. A characteristic finding strongly suggestive of mitochondrial damage is a very high ratio of lactic acid to pyruvic acid (lactate to pyruvate). Pyruvate is the salt of pyruvic acid and is the core product of glycolysis. In heavy exercise, glycolysis is increased many times for additional energy, resulting in increased pyruvate synthesis. However, the excess of pyruvate can not be fully processed in the citric acid cycle so that the excess pyruvate is converted to lactate. Thus, a reduced pyruvate content in the blood is usually associated with degradation disorders in the carbohydrate metabolism. The procedure for the blood analysis does not differ from those of the above-mentioned examples in the examination of the lactate / pyruvate ratio. For this examination as well, the test person has to take approximately two milliliters of bodily fluid (blood) in a first step, which is then transferred to a measuring cuvette 6 and cooled to 200 K to 10 K within the measuring device 1. In the following, the frozen sample 5 is irradiated with light and the resulting fluorescence is picked up by the measuring system. The fact that the fluorescence is detectable as a function of the reduction in temperature in the case of the lactate has already been shown in the example of the determination of the lactate value. The Fig. 5 shows an example of the fluorescence emission of pyruvate under different temperature conditions. For the experiment shown here, sample 5 was excited to fluoresce with laser light of wavelength 266 nm. It can be seen very well here that pyruvate under these conditions did not show any fluorescence even at 250 K. A first evaluable spectrum could be measured at about 200 K within these experiments. A further reduction in temperature also led to increases in the fluorescence intensity and better resolutions in the spectra structure in the case of pyruvates. From a temperature of 125 K on these investigations, a spectrum could be detected, which could not be significantly improved even with further reduction of the temperature. The fluorescence data of lactate and pyruvate obtained in this way are normalized and their characteristic data, for example the position of the band maxima, the intensity of the fluorescence, the ratio of different band maxima to each other, the lifetime of the fluorescence, compared with tabulated values of the subjects before the load and evaluated. In addition, the obtained fluorescence data of pyruvate and lactate in a further step for the evaluation are set in relation to each other.

In summary, it can be shown for all three of the application examples described here that the method described brings a clear knowledge advantage when used in these examination fields. It also shows that all three examinations could be performed by one method. Since the selected examples are all based, at least in part, on data obtained from a blood analysis, this method even makes it theoretically possible to conduct these examinations virtually parallel to a body fluid sample for a subject. The investigation of further indicators from body fluids, which point to other clinical pictures, is also conceivable with this method. Here, however, as already mentioned, care must be taken that not all of the selected indicators are optimally stimulated to fluoresce at the same wavelength. Thus, care must be taken in the design of the measuring device 1 to include some different light sources in the measurement process.

LIST OF REFERENCE SIGNS

1

measuring device

2

light source

3

cryostat

4

Fluorescence spectrograph, imaging detector

5

Sample, body fluid sample, sample components, blood sample

6

Cuvette, measuring cuvette

7

Sample cell, sample holder

8th

Heat exchanger

9

heater

10

Kryostatenwandung

11

UV-standard windows

12

compressors

13

hose system

14

Computer, evaluation unit

Claims (15)

Method for the qualitative and quantitative analysis of body fluids, in particular of blood, urine or sweat, using fluorescence spectroscopic methods for the examination of bodily fluids, with method steps a) to e): a) temperature control of the body fluid sample (5) at temperatures between 200 K and 4 K in a cryostat (3); b) Irradiation and excitation of the body fluid sample (5) with light in the UV, visible or infrared range at temperatures between 200 K and 4 K, wherein the cryostat (3) both the function of the temperature control of the body fluid sample (5) and the function of a Sample holder (7) and a sample measuring cell (7) fulfilled by the sample measuring cell (7) in the cryostat (3) and is closed by a Kryostatenwandung (10) to the outside and thereby directly with a heat exchanger (8) of the cryostat (3) thermally contacted; c) spectral decomposition of the resulting fluorescence radiation of the body fluid sample (5) into individual fluorescence signals; d) Detecting the fluorescence signals and e) Comparison of the fluorescence signals obtained and of corresponding comparative data and evaluation. A method according to claim 1, characterized in that the temperature of the body fluid sample (5) in process step a) takes place at temperatures between 150 K and 4 K. Method according to one of claim 1 or 2, characterized in that the irradiation of the body fluid sample (5) takes place in step b) with several different wavelengths partly also simultaneously. Method according to one of claims 1 to 3, characterized in that the detection according to process step d) of the fluorescence signals from different components of the body fluid sample (5) takes place simultaneously. A method according to claim 4, characterized in that in the analysis of blood components at least two of the indicators blood sugar levels, insulin levels, levels of lipoproteins, levels of homocysteine, long-term blood sugar (HbA1 c), the content of fibrinogen and blood protein values are measured simultaneously, wherein in the determination the lactate / pyruvate ratio, the measurements of the fluorescence signals of lactate and pyruvate take place simultaneously. A method according to claim 4, characterized in that carried out in the analysis of urine components, the measurements of the sugar content, the content of acetone and / or albumin. Method according to one of claims 1 to 6, characterized in that for the comparison of the fluorescence signals obtained and the comparison data in step e) as comparison characteristics, the intensity of fluorescence, the area below a fluorescence band, the position of the individual fluorescence band maxima in the total spectrum, the ratio of different Band maxima within a spectrum to each other, the quantum yield of the body fluid sample (5) and the Lifespan of the fluorescence of the individual fluorophores are provided. Measuring device for carrying out the method according to one of claims 1 to 7, comprising A light source (2) for the stimulation of the body fluid sample (5), A cryostat (3) for tempering the body fluid sample (5), A fluorescence spectrograph (4) for decomposing the fluorescence radiation of the body fluid sample (5) obtained, An imaging detector (4), A sample measuring cell (7), which simultaneously serves as sample holder (7) and is arranged in the cryostat (3), being closed to the outside through a cryostat wall (10) and directly connected to a heat exchanger (8) of the cryostat (3) thermally contacted, and • A UV-compatible, temperature-stable cuvette (6), with which the body fluid sample (5) is introduced into the sample cell (7). Measuring device according to claim 8, characterized in that the light source (2), the cryostat (3) for tempering the body fluid sample (5), the fluorescence spectrograph (4) and the imaging detector (4) within a compact measuring device (1) are arranged. Measuring device according to claim 8 or 9, characterized in that the Kryostatenwandung (10), which closes the sample measuring cell (7) towards the outside, two 90 ° offset from each other UV-common window (11). Measuring device according to one of claims 8 to 10, characterized in that the light source (2) which is used for the excitation of the frozen body fluid sample (5), as a broadband light source, in particular in the form of a mercury vapor lamp, a high-pressure xenon lamp or a Deuterium lamp, is formed. Measuring device according to one of claims 8 to 10, characterized in that the light source (2), which is used for the excitation of the frozen body fluid sample (5), is designed as a narrow-band light source. Measuring device according to claim 12, characterized in that in the measuring device a plurality of light sources (2) of different wavelengths are provided. Measuring device according to one of claims 8 to 13, characterized in that the cryostat (3) has a closed refrigerant circuit, being provided as a refrigerant helium, nitrogen or refrigerant mixtures. Measuring device according to one of claims 8 to 14, characterized in that the sample measuring cell (7) directly thermally contacted with a heater (9) for regulating the temperature of the body fluid sample (5).

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